Oxidative damage to DNA induced by reactive oxygen species (ROS) has been linked to carcinogenesis and is believed to play a role in aging. The most common lesion, 8-oxoguanine (8-oxoG), is known to lead to G:C to A:T transversion mutations, the most frequent mutation found in cancers. In humans, the base excision repair (BER) enzyme 8-oxoguanine DNA glycosylase (hOGG1) is responsible for locating and repairing these rare 8- oxoG lesions amongst billions of undamaged base pairs. It is widely accepted that this feat is accomplished via two modes of facilitated diffusion: a 1D mode involving a loosely associated enzyme capable of sliding along the DNA and a 3D mode that involves intermittent dissociation and re-association of the enzyme with the DNA chain (hopping). As most studies have involved the use of dilute, low-salt solutions, a key unknown is how these search states respond to the high ionic strength and a crowded environment that is present in the human cell nucleus. The focus of this proposal is to fully characterize the DNA damage search and repair pathway under physiologically relevant conditions and determine general principles for damage repair through a comparative study using hOGG1 and human uracil DNA glycosylase (hUNG). These are representatives of two of the largest DNA glycosylase super families. In the first aim, an extensive array of thermodynamic and kinetic experiments involving fluorescence anisotropy, steady-state kinetics, stopped-flow kinetics, and the molecular clock intermolecular site transfer method will be used to probe how microscopic steps of the hOGG1 damage search pathway respond to the key solution variable ionic strength, molecular crowding and high densities of non-specific decoy DNA. These results will be compared with those obtained with hUNG in order to evaluate common responses to these solution variables. Preliminary data has already established that molecular crowding produces a dramatic enhancement of the sliding pathway and that physiological concentrations of salt enhance DNA damage specificity. The second aim will evaluate the comprehensive mechanism for locating and repairing 8-oxoG lesions in the human cell nucleus for the first time. We have developed an innovative strategy that allows for the investigation of facilitated diffusion and the probability of hOGG1 and hUNG to repair of multiple lesion sites as a function of site spacing in human cells. Rigorous interpretation of the in cell findings will be facilitated by the highly-controlled in vitro studies.